Thioadenosine Derivatives as Species-Independent A3 Adenosine Rece

Sep 24, 2008 - Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans .... derivatives 7 as highly potent and species-independent A3 AR...
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J. Med. Chem. 2008, 51, 6609–6613

6609

Structure-Activity Relationships of Truncated D- and L-4′-Thioadenosine Derivatives as Species-Independent A3 Adenosine Receptor Antagonists1 Lak Shin Jeong,*,† Shantanu Pal,† Seung Ah Choe,† Won Jun Choi,† Kenneth A. Jacobson,‡ Zhan-Guo Gao,‡ Athena M. Klutz,‡ Xiyan Hou,† Hea Ok Kim,† Hyuk Woo Lee,† Sang Kook Lee,† Dilip K. Tosh,† and Hyung Ryong Moon§ Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans UniVersity, Seoul 120-750, Korea, Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes & DigestiVe & Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, College of Pharmacy, Pusan National UniVersity, Busan 609-753, Korea ReceiVed July 14, 2008

Novel D- and L-4′-thioadenosine derivatives lacking the 4′-hydroxymethyl moiety were synthesized, starting from D-mannose and D-gulonic γ-lactone, respectively, as potent and selective species-independent A3 adenosine receptor (AR) antagonists. Among the novel 4′-truncated 2-H nucleosides tested, a N6-(3chlorobenzyl) derivative 7c was the most potent at the human A3 AR (Ki ) 1.5 nM), but a N6-(3-bromobenzyl) derivative 7d showed the optimal species-independent binding affinity. Chart 1. Rationale for the Design of the Target Nucleosides 7

Introduction On the basis of the structure of adenosine, an endogenous cell signaling molecule that binds to four specific subtypes (A1, A2A, A2B, and A3) of adenosine receptors (ARsa),2 a number of nucleoside analogues have been synthesized and evaluated as adenosine receptor ligands.3 Among these, IB-MECA4 1 and Cl-IB-MECA5 2 were discovered as potent and selective A3 AR full agonists (Ki ) 1.0 and 1.4 nM, respectively, at the human A3 AR) and are being developed as anti-inflammatory and anticancer agents. On the basis of the bioisosteric rationale, we reported the 4′-thionucleosides 3 and 4, derivatives of compounds 1 and 2, to also be highly potent and selective A3 AR full agonists.6 Compound 4 exhibited potent in vitro and in vivo antitumor activities,7 resulting from the inhibition of Wnt signaling pathway (Chart 1). However, because of the structural similarity to adenosine, most of these adenosine analogues were found to be A3 AR agonists. Only a few nucleoside derivatives8 have been reported to be A3 AR antagonists, but these generally exhibit weaker and less selective human A3 AR antagonism than nonpurine heterocyclic A3 AR antagonists. Although these nonpurine heterocyclic A3 AR antagonists9 bound with high affinity at the human A3 AR, they were weak or ineffective at the rat A3 AR, indicating that they were not ideal for evaluation in small animal models and thus as drug candidates.10 Therefore, it is highly desirable to develop A3 AR antagonists that are independent of species. The fact10 that nucleoside analogues show minimal species-dependence at the A3 AR prompted us to search for novel potent and selective A3 AR antagonists, derived from nucleoside templates. * To whom correspondence should be addressed. Phone: 82-2-3277-3466. Fax: 82-2-3277-2851. E-mail: [email protected]. † Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University. ‡ Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health. § College of Pharmacy, Pusan National University. a Abbreviations: AR, adenosine receptor; CCPA, 2-chloro-N6-cyclopentyladenosine; CHO, Chinese hamster ovary; IB-MECA, N6-(3-iodobenzyl)5′-N-methylcarboxamidoadenosine; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)5′-N-methylcarboxamidoadenosine; I-AB-MECA, N6-(4-amino-3-iodobenzyl)5′-N-methylcarboxam idoadenosine; NECA, 5′-N-ethylcarboxamidoadenosine.

A molecular modeling study of the A3 AR indicated that hydrogen of the 5′-uronamides of compounds 1-4 serves as a hydrogen-bonding donor in the binding site of the A3 AR, which is essential for the induced-fit required for the activation of the A3 AR.11 On the basis of these findings, we appended extra alkyl groups on the 5′-uronamides of compounds 1-4 to remove hydrogen-bonding ability at this site, thus precluding the conformational change required for activation of the A3 AR. As expected, these 5′-N,N-dialkyl amide derivatives12 displayed potent and selective A3 AR antagonism in which steric factors were crucial for affinity in binding to the A3 AR. Within this class, 5′-N,N-dimethylamide derivative 5 was discovered to be the most potent full A3 AR antagonist. Encouraged with these results, we designed and synthesized another new template to remove the 5′-uronamide group of compound 4 in order to minimize the steric repulsion at the binding region of the 5′uronamide group and to abolish its hydrogen-bonding ability. This led to the discovery of compounds 6a-6e as highly potent and selective human A3 AR antagonists, which were more potent and selective than compound 5.1 Among these, compound 6e

10.1021/jm8008647 CCC: $40.75  2008 American Chemical Society Published on Web 09/24/2008

6610 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20

Scheme 1. Synthesis of Truncated D-4′-Thioadenosine Derivatives 7a-7ea

a Reagents and conditions: (a) 6-chloropurine, ammonium sulfate, HMDS, 170 °C, 15 h, then TMSOTf, DCE, rt to 80 °C, 3 h; (b) 2 N HCl, 45 °C, 15 h; (c) RNH2, Et3N, EtOH, rt, 1-3 d.

also showed species-independent binding affinity, as indicated by its high affinity at the rat A3 AR.1 On the basis of these results, it is of interest to systematically establish structure-activity relationships by modifying the C2 and N6 positions of the purine moiety of compounds 6a-6e in order to develop novel A3 AR antagonists. In this article, we extend previous observations that truncated D-4′-thioadenosine derivatives 6a-6e containing 2-Cl substitution are selective A3 AR antagonists.1 A series of 2-H analogues were prepared and characterized biologically. The binding affinities at the human A3 AR were compared with those at the rat A3 AR to develop species-independent A3 AR antagonists. We also compared the binding affinities of D-4′thionucleosides with those of the corresponding L-4′-thionucleosides to determine a stereochemical preference. Thus, here we report a full account of truncated D- and L-4′-thioadenosine derivatives 7 as highly potent and species-independent A3 AR antagonists. Results and Discussion The D-glycosyl donor 8 was subjected to the Lewis acidcatalyzed condensation for the synthesis of the final D-4′thionucleosides lacking a 4′-hydroxymethyl group, as shown in Scheme 1. The D-glycosyl donor 8 was condensed with 6-chloropurine in the presence of TMSOTf as a Lewis acid to give β-6-chloropurine derivative 9 as a single diastereomer. The anomeric configuration of compound 9 was easily confirmed by 1H NOE experiment between 3′-H and H-8. Removal of the isopropylidene group of 9 was achieved with 2 N HCl in THF to give 10. The 2-H intermediate 10 was converted to the novel N6-methyl derivative 7a and N6-3-halobenzyl derivatives 7b-7e by treating with methylamine and 3-halobenzylamines, respectively. This route parallels the synthesis of the 2-chloro-N6susbtituted-4′-thiopurine analogues 6a-6e that we reported earlier.1

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Scheme 2. Synthesis of Truncated L-4′-Thioadenosine Derivatives 7f and 7g

To determine whether a stereochemical preference exists in the binding to the A3 AR, the L-enantiomers 7f and 7g of D-4′thionucleosides were synthesized as illustrated in Scheme 2. D-Gulonic γ-lactone was converted to the diol 11 according to our previously published procedure.13 One-step conversion of the diol 11 into the L-glycosyl donor 12 was achieved using excess Pb(OAc)4, indicating that oxidative diol cleavage, oxidation of the resulting aldehyde to the acid, and oxidative decarboxylation occurred simultaneously.13 Using the same synthetic strategy shown in Scheme 1, L-4′-thioadenosine derivatives 7f and 7g were synthesized from L-glycosyl donor 12. Initial binding experiments were performed using adherent mammalian cells stably transfected with cDNA encoding the appropriate human ARs (A1 AR and A3 AR in CHO cells and A2A AR in HEK-293 cells).14,15 Binding was carried out using 1 nM [3H]CCPA, 10 nM [3H]CGS-21680, or 0.5 nM [125I]IAB-MECA as radioligands for A1, A2A, and A3 ARs, respectively. As shown in Table 1, most of the synthesized compounds exhibited high binding affinity at the human A3 AR with low binding affinities at the human A1 AR and human A2A AR. Among the novel 2-H truncated adenosine derivatives tested, compound 7c (R ) 3-chlorobenzyl) showed the highest binding affinity (Ki ) 1.5 ( 0.4 nM) at the human A3 AR with high selectivities versus the A1 AR (570-fold selective) and the A2A AR (290-fold selective). Compound 7e (R ) 3-iodobenzyl) was also very potent (Ki ) 2.5 ( 1.0 nM), with selectivities of 210and 92-fold versus the A1 and A2A AR, respectively. N6Substituted adenosine derivatives 7a-7e without a 2-chloro substituent showed a very similar pattern to the corresponding 2-chloro derivatives 6a-6e in the binding affinity at the human A3 AR but showed less selectivity versus the other subtypes of ARs. In the 3-halobenzyl series, the order of binding affinity for 2-H analogues was as follows: Cl > I > Br > F, indicating that the size of halogen alone does not determine the binding affinity at the human A3 AR. It is interesting to note that 2-H derivatives are less lipophilic than the corresponding 2-Cl derivatives, conferring more water solubility on the molecules for further biological evaluation. For example, the cLogP values of corresponding structures 6c and 7c are 1.84 and 1.12, respectively. To determine a stereochemical preference, the binding affinities of D-series were compared with those of L-series. As shown in Table 1, L-type nucleosides 7f and 7g

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Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6611

Table 1. Binding Affinities of Known A3 AR Agonists, 1-4 and Antagonist 5, and Truncated 4′-Thioadenosine Derivatives 6a-6e and 7a-7g at Three Subtypes of ARs

affinity, Ki, nM ( SEM (or % inhibition at 10-5 M)a,b compd

hA1

hA2A

rA3

hA3c

1(IB-MECA) 2 (Cl-IB-MECA) 3 (thio-IB-MECA) 4 (thio-Cl-IB-MECA) 5 6a (R1 ) Cl, R2 ) methyl) 6b (R1 ) Cl, R2 ) 3-fluorobenzyl) 6c (R1 ) Cl, R2 ) 3-chlorobenzyl) 6d (R1 ) Cl, R2 ) 3-bromobenzyl) 6ed (R1 ) Cl, R2 ) 3-iodobenzyl) 7a (R1 ) H, R2 ) methyl) 7b (R1 ) H, R2 ) 3-fluorobenzyl) 7c (R1 ) H, R2 ) 3-chlorobenzyl) 7d (R1 ) H, R2 ) 3-bromobenzyl) 7e (R1 ) H, R2 ) 3-iodobenzyl) 7f (R1 ) Cl, R2 ) 3-bromobenzyl) 7g (R1 ) Cl, R2 ) 3-iodobenzyl)

51 222 ( 22 17.3 193 ( 46 6220 ( 640 55.4 ( 1.8 (20%) (38%) (34%) 2490 ( 940 1070 ( 180 1430 ( 420 860 ( 210 790 ( 190 530 ( 97 (6.1%) (-8.0%)

2900 5360 ( 2470 ND 223 ( 36 >10,000 45.0 ( 1.4 (48%) (18%) (18%) 341 ( 75 (22 ( 5%) 1260 ( 330 440 ( 110 420 ( 32 230 ( 65 (45.7%) (-0.95%)

1.1 0.33 1.86 ( 0.36 0.82 ( 0.27 321 ( 74 658 ( 160 36.2 ( 10.7 6.2 ( 1.8 6.1 ( 1.8 3.89 ( 1.15 (28 ( 10%) 98 ( 28 17 ( 5 6.3 ( 1.3 48 ( 7 ND ND

1.0 1.4 ( 0.3 0.25 ( 0.06 0.38 ( 0.07 15.5 ( 3.1 3.69 ( 0.25 7.4 ( 1.3 1.66 ( 0.90 8.99 ( 5.17 4.16 ( 0.50 4.8 ( 1.7 7.3 ( 0.6 1.5 ( 0.4 6.8 ( 3.4 2.5 ( 1.0 (12.6%) (18.4%)

a ND: Not determined. All binding experiments were performed using adherent mammalian cells stably transfected with cDNA encoding the appropriate human AR (A1 AR and A3 AR in CHO cells and A2A AR in HEK-293 cells) or the rat A3 AR (CHO cells). Binding was carried out using 1 nM [3H]CCPA, 10 nM [3H]CGS-21680, or 0.5 nM [125I]I-AB-MECA as radioligands for A1, A2A, and A3 ARs, respectively. Values are expressed as mean ( sem, n ) 3-4 (outliers eliminated), and normalized against a nonspecific binder, 5′-N-ethylcarboxamidoadenosine (NECA, 10 µM). Data for compounds 6a-6e at the human ARs and compound 6e at the rat A3 AR were reported in ref 1. b A value expressed as a percentage refers to percent inhibition of specific radioligand binding at 10 µM, with nonspecific binding defined using 10 µM NECA. c A functional assay was also carried out at this subtype: percent inhibition at 10 µM forskolin-stimulated cyclic AMP production in CHO cells expressing the human A3 AR, as a mean percentage of the response of the full agonist 3 (n ) 1 - 3). None of the analogues 5-7 activated the hA3 AR (>10% of full agonist effect) by this criterion. d Compound 6e at 10 µM displayed Br ≈ Cl > F > Me. The 2-Cl derivatives generally showed more potent and species-independent binding affinity than the corresponding 2-H analogues. Compound 6e exhibited the highest binding affinity at the rat A3 AR (Ki ) 3.89 ( 1.15 nM) among all compounds tested and was inactive as agonist or antagonist in a cyclic AMP functional assay16,17 at the hA2B AR. It is interesting to note that N6-methyl derivatives 6a and 7a showing high binding affinities (Ki ) 3.69 ( 0.25 nM and 4.8 ( 1.7 nM, respectively) at the human A3 AR lost their binding affinities at the rat A3 AR, indicating that there must be a larger N6 substituent for species-independent binding affinity at the A3 AR.18,19

In a functional assay, percent inhibition at 10 µM forskolinstimulated cyclic AMP production in CHO cells expressing the human A3 AR was measured as a mean percentage of the response of the full agonist 3 (n ) 1 - 3). None of the analogues 6 and 7 activated the human A3 AR (>10% of full agonist effect) by this criterion. Conclusion We have established structure-activity relationships of novel truncated D- and L-4′-thionucleoside analogues as potent speciesindependent A3 AR antagonists. The glycosyl donors 8 and 12 were efficiently synthesized from D-mannose and D-gulonic γ-lactone, respectively, using ring closure of dimesylate with sodium sulfide and one step conversion of the diol into the acetate with lead tetraacetate as key steps. Among the novel 4′-truncated 2-H nucleosides tested, D-N6-(3-halobenzyl) derivatives 7b-7e exhibited high binding affinities at the human A3 AR as well as at the rat A3 AR with very low binding affinities at the human A1 and A2A ARs and a N6-(3-chlorobenzyl) derivative 7c was the most potent at the human A3 AR, but at the rat A3 AR 3-bromobenzyl derivative, 7d was the most potent. Among both 2-H and 2-Cl analogues tested, 2-chloro-N6-(3iodobenzyl) derivative 6e was found to exhibit the most potent binding affinity at the rat A3 AR. Because this class of potent nucleoside human A3 AR antagonists showed species-independence in interaction at this AR subtype, they are regarded as

6612 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20

good candidates for efficacy evaluation in small animal models and for further drug development. Experimental Section General Methods. Melting points are uncorrected. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were measured in CDCl3, CD3OD or DMSO-d6, and chemical shifts are reported in parts per million (δ) downfield from tetramethylsilane as internal standard. Column chromatography was performed using silica gel 60 (230-400 mesh). Anhydrous solvents were purified by the standard procedures. cLogP values were calculated using ChemDrawUltra, version 11.0 (CambridgeSoft). Synthesis. 6-Chloro-9-((3aR,4R,6aS)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-9H-purine (9). 6-Chloropurine (3.91 g, 25.3 mmol), ammonium sulfate (84 mg, 0.63 mmol), and HMDS (50 mL) were refluxed under inert and dry conditions overnight. The solution was evaporated under high vacuum. The resulting solid was redissolved in 1,2-dichloroethane (20 mL) cooled in ice. The solution of 81 (2.76 g, 12.6 mmol) in 1,2-dichloroethane (20 mL) was added to this mixture dropwise. TMSOTf (4.6 mL, 25.3 mmol) was added dropwise to the mixture. The mixture was stirred at 0 °C for 30 min, at rt for 1 h, and then heated at 80 °C for 2 h. The mixture was cooled, diluted with CH2Cl2, and washed with saturated NaHCO3 solution. The organic layer was dried with anhydrous MgSO4 and evaporated under reduced pressure. The yellowish syrup was subjected to a flash silica gel column chromatography (CH2Cl2:MeOH ) 50:1) to give 9 (3.59 g, 90%) as a foam: [R]23.6D -157.63 (c 0.144, DMSO); FAB-MS m/z 313 [M + H]+; UV (MeOH) λmax 265.0 nm. 1H NMR (CDCl3) δ 8.67 (s, 1 H), 8.23 (s, 1 H), 5.88 (s, 1 H), 5.25-5.19 (m, 1 H), 3.69 (dd, 1 H, J ) 4.0, 13.2 Hz), 3.18 (d, 1 H, J ) 12.8 Hz), 1.51 (s, 3 H), 1.28 (s, 3 H). 13C NMR (CDCl3) δ 152.0, 151.4, 151.1, 144.3, 132.6, 111.9, 89.6, 84.3, 70.3, 40.8, 26.4, 24.6. Anal. (C12H13ClN4O2S) C, H, N, S. (2R,3R,4S)-2-(6-Chloro-9H-purin-9-yl)-tetrahydrothiophene3,4-diol (10). Hydrochloric acid (2 N, 12 mL) was added to a solution of 9 (2.59 g, 8.28 mmol) in THF (20 mL), and the mixture was stirred at room temperature overnight. The mixture was neutralized with 1 N NaOH solution, and then the volatiles were carefully evaporated under reduced pressure. The mixture was subjected to a flash silica gel column chromatography (CH2Cl2: MeOH ) 20:1) to give 10 (1.79 g, 79%) as a white solid: [R]23.5D -109.14 (c 0.164, DMSO); FAB-MS m/z 273 [M + H]+; mp 192.3-192.8 °C; UV (MeOH) λmax 264.5 nm. 1H NMR (DMSOd6) δ 9.02 (s, 1 H), 8.81 (s, 1 H), 6.02 (d, 1 H, J ) 7.2 Hz), 5.62 (d, 1 H, J ) 6.0 Hz, D2O exchangeable), 5.43 (d, 1 H, J ) 4.1 Hz, D2O exchangeable), 4.74-4.70 (m, 1 H), 4.40-4.36 (m, 1 H), 3.47 (dd, 1 H, J ) 4.0, 11.2 Hz), 2.83 (dd, 1 H, J ) 2.8, 11.2 Hz). 13C NMR (DMSO-d6) 152.1, 151.6, 149.2, 146.6, 131.3, 78.6, 72.1, 62.4, 34.7. Anal. (C9H9ClN4O2S) C, H, N, S. General Procedure for the Synthesis of 7a-7e. To a solution of 10 in EtOH (5 mL) was added appropriate amine (1.5 equiv) at room temperature, and the mixture was stirred at rt for a time period ranging from 2 h to 3 d and evaporated. The residue was purified by a flash silica gel column chromatography (CH2Cl2:MeOH ) 20:1) to give 7a-7e. (2R,3R,4S)-Tetrahydro-2-(6-(methylamino)-9H-purin-9-yl)thiophene-3,4-diol (7a). Yield 83%; [R]22.8D -175.60 (c 0.123, DMSO); FAB-MS m/z 268 [M + H]+; mp 223.9-224.8 °C; UV (MeOH) λmax 266.0 nm. 1H NMR (DMSO-d6) δ 8.40 (s, 1 H), 8.23 (s, 1 H), 7.72 (br s, 1 H, D2O exchangeable), 5.89 (d, 1 H, J ) 7.2 Hz), 5.51 (d, 1 H, J ) 6.4 Hz, D2O exchangeable), 5.32 (d, 1 H, J ) 4.4 Hz, D2O exchangeable), 4.70-4.64 (m, 1 H), 4.37-4.33 (m, 1 H), 3.40 (dd, 1 H, J ) 4.0, 10.8 Hz), 2.95 (s, 3 H), 2.79 (dd, 1 H, J ) 3.2, 10.8 Hz). 13C NMR (DMSO-d6) δ 154.9, 152.5, 148.8, 139.5, 119.5, 78.3, 72.2, 61.5, 34.6, 27.0. Anal. (C10H13N5O2S) C, H, N, S. (2R,3R,4S)-2-(6-(3-Fluorobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (7b). Yield 82%; [R]23.7D -141.22 (c 0.114, DMSO); FAB-MS m/z 362 [M + H]+; mp 180.5-180.7

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°C; UV (MeOH) λmax 273.5 nm; 1H NMR (DMSO-d6) δ 8.45 (s, 1 H), 8.43 (br s, 1 H, D2O exchangeable), 8.21 (s, 1 H), 7.36-7.30 (m, 1 H), 7.18-7.11 (m, 2 H), 7.03 (dt, 1 H, J ) 2.4, 8.4 Hz), 5.90 (d, 1 H, J ) 7.2 Hz), 5.53 (d, 1 H, J ) 6.4 Hz, D2O exchangeable), 5.35 (d, 1 H, J ) 4.0 Hz, D2O exchangeable), 4.70-4.66 (m, 2 H), 4.36-4.33 (m, 1 H), 3.41 (dd, 1 H, J ) 4.0, 10.8 Hz), 3.17 (d, 1 H, J ) 5.2 Hz), 2.79 (dd, 1 H, J ) 2.8, 10.8 Hz). 13C NMR (DMSO-d6) δ 163.4, 160.9, 152.4, 143.2, 140.0, 130.2, 130.1, 123.1, 123.1, 113.8, 113.6, 113.4, 113.2, 78.3, 72.2, 61.6, 48.6, 34.4. Anal. (C16H16FN5O2S) C, H, N, S. (2R,3R,4S)-2-(6-(3-Chlorobenzylamino)-9H-purin-9-yl)-tetrahydrothiophene-3,4-diol (7c). Yield 85%; [R]23.9D -162.5 (c 0.096, DMSO); FAB-MS m/z 378 [M + H]+; mp 165.0-165.3 °C; UV (MeOH) λmax 274.5 nm. 1H NMR (DMSO-d6) δ 8.46 (s, 1 H), 8.44 (br s, 1 H, D2O exchangeable), 8.22 (s, 1 H), 7.39-7.24 (m, 4 H), 5.90 (d, 1 H, J ) 10.4 Hz), 5.53 (d, 1 H, J ) 6.4 Hz, D2O exchangeable), 5.35 (d, 1 H, J ) 4.0 Hz, D2O exchangeable), 4.71-4.67 (m, 2 H), 4.38-4.33 (m, 1 H), 3.47-3.31 (m, 2 H), 2.80 (dd, 1 H, J ) 3.2, 10.8 Hz). 13C NMR (DMSO-d6) δ 154.3, 152.4, 142.8, 140.0, 132.8, 130.1, 126.9, 126.6, 125.8, 78.3, 72.2, 61.6, 56.0, 34.4. Anal. (C16H16ClN5O2S) C, H, N, S. (2R,3R,4S)-2-(6-(3-Bromobenzylamino)-9H-purin-9-yl)-tetrahydrothiophene-3,4-diol (7d). Yield 71%; [R]23.7D -100.71 (c 0.139, DMSO); FAB-MS m/z 422 [M]+; mp 183.0-184.0 °C; UV (MeOH) λmax 270.0 nm. 1H NMR (DMSO-d6) δ 8.46 (s, 1 H), 8.43 (br s, 1 H, D2O exchangeable), 8.21 (s, 1 H), 7.53 (s, 1 H) 7.42-7.24 (m, 3 H), 5.90 (d, 1 H, J ) 7.2 Hz), 5.53 (d, 1 H, J ) 6.4 Hz, D2O exchangeable), 5.35 (d, 1 H, J ) 4.0 Hz, D2O exchangeable), 4.71-4.66 (m, 2 H), 4.37-4.34 (m, 1 H), 3.41 (dd, 1 H, J ) 4.0, 10.8 Hz), 3.06 (q, 1 H, J ) 7.2 Hz). 2.79 (dd, 1 H, J ) 2.8, 10.8 Hz). 13C NMR (DMSO-d6) δ 154.2, 152.4, 143.0, 140.0, 130.4, 129.8, 129.4, 126.2, 121.5, 78.3, 72.2, 61.6, 45.5, 34.5. Anal. (C16H16BrN5O2S) C, H, N, S. (2R,3R,4S)-2-(6-(3-Iodobenzylamino)-9H-purin-9-yl)-tetrahydrothiophene-3,4-diol (7e). Yield 88%; [R]23.8D -97.08 (c 0.137, DMSO); FAB-MS m/z 370 [M + H]+; mp 198.8-199.8 °C; UV (MeOH) λmax 271.5 nm. 1H NMR (DMSO-d6) δ 8.45 (s, 1 H), 8.43 (br s, 1 H, D2O exchangeable), 8.21 (s, 1 H), 7.72 (s, 1 H), 7.56 (d, 1 H, J ) 7.2 Hz), 7.35 (d, 1 H, J ) 7.6 Hz), 7.10 (merged dd, 1 H, J ) 7.6 Hz), 5.90 (d, 1 H, J ) 7.2 Hz), 5.53 (d, 1 H, J ) 6.4 Hz, D2O exchangeable), 5.35 (d, 1 H, J ) 4.4 Hz, D2O exchangeable), 4.71-4.66 (m, 2 H), 4.37-4.34 (m, 1 H), 3.41 (dd, 1 H, J ) 2.8, 10.8 Hz), 3.15 (d, 1 H, J ) 5.2 Hz), 2.79 (dd, 1 H, J ) 2.8, 10.8 Hz). 13C NMR (DMSO-d6) δ 154.2, 152.4, 149.2, 142.9, 140.0, 137.0, 135.7, 135.3, 130.4, 126.6, 94.7, 78.3, 72.2, 61.6, 42.2, 34.4. Anal. (C16H16IN5O2S) C, H, N, S. L-4-Thiosugar acetate 12 was synthesized from D-gulonic acid γ-lactone according to a similar procedure1,13 used for the preparation of 8 (Scheme 1). Then L-4-thiosugar acetate 12 was converted to 13 according to a similar procedure used for the preparation of 9. The final L-4′-thio nucleosides 7f and 7g were synthesized from 12 according to the described general procedure for the synthesis of 7a-7e. The 1H, 13C NMR, UV, and mp data of L-series compounds were the same as for the D-series of compounds as described above, except that the specific optical rotations were in the opposite direction. Yields of the L-series compounds were comparable with those of the D-series of compounds. Binding Assays.1,6 Human A1 and A2A ARs. For binding to human A1 AR, [3H]CCPA (1 nM) was incubated with membranes (40 µg/tube) from CHO cells stably expressing human A1 ARs at 25 °C for 60 min in 50 mM Tris · HCl buffer (pH 7.4; MgCl2, 10 mM) in a total assay volume of 200 µL. Nonspecific binding was determined using 10 µM of NECA. For human A2A AR binding, membranes (20 µg/tube) from HEK-293 cells stably expressing human A2A ARs were incubated with 15 nM [3H]CGS21680 at 25 °C for 60 min in 200 µL 50 mM Tris · HCl, pH 7.4, containing 10 mM MgCl2. NECA (10 µM) was used to define nonspecific binding. Reaction was terminated by filtration with GF/B filters. Human and Rat A3 ARs. For competitive binding assay, each tube contained 100 µL of membrane suspension (from CHO cells

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stably expressing the human or rat A3 AR, 20 µg protein), 50 µL of [125I]I-AB-MECA (0.5 nM), and 50 µL of increasing concentrations of the nucleoside derivative in Tris · HCl buffer (50 mM, pH 7.4) containing 10 mM MgCl2. Nonspecific binding was determined using 10 µM of NECA in the buffer. The mixtures were incubated at 25 °C for 60 min. Binding reactions were terminated by filtration through Whatman GF/B filters under reduced pressure using a MT24 cell harvester (Brandell, Gaithersburg, MD). Filters were washed three times with 9 mL of ice-cold buffer. Radioactivity was determined in a Beckman 5500B γ-counter. For binding at all three subtypes, Ki values are expressed as mean ( sem, n ) 3-4 (outliers eliminated), and normalized against a nonspecific binder, 5′-N-ethylcarboxamidoadenosine (NECA, 10 µM). Alternately, for weak binding, a percent inhibition of specific radioligand binding at 10 µM, relative to inhibition by 10 µM NECA assigned as 100%, is given.

Acknowledgment. This work was supported by the Korea Research Foundation grant (KRF-2008-E00304) and the Intramural Research Program of NIDDK, NIH, Bethesda, MD. Supporting Information Available: Elemental analyses data for all unknown compounds and pharmacological methods. This material is available free of charge via the Internet at http:// pubs.acs.org.

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